Histopathological effects of long-term exposure to realistic concentrations of cadmium in the hepatopancreas of Sparus aurata juveniles.

In recent decades, cadmium has emerged as an environmental stressor in aquatic ecosystems due to its persistence and toxicity. It can enter water bodies from various natural and anthropogenic sources and, once introduced into aquatic systems, can accumulate in sediments and biota, leading to bioaccumulation and biomagnification in the food chain. For this reason, the effects of cadmium on aquatic life remain an area of ongoing research and concern. In this paper, a multidisciplinary approach was used to assess the effects of long-term exposure to an environmental concentration on the hepatopancreas of farmed juveniles of sea bream, Sparus aurata. After determining metal uptake, metallothionein production was assessed to gain insight into the organism's defence response. The effects were also assessed by histological and ultrastructural analyses. The results indicate that cadmium accumulates in the hepatopancreas at significant concentrations, inducing structural and functional damage. Despite the parallel increase in metallothioneins, fibrosis, alterations in carbohydrate distribution and endocrine disruption were also observed. These effects would decrease animal fitness although it did not translate into high mortality or reduced growth. This could depend on the fact that the animals were farmed, protected from the pressure deriving from having to search for food or escape from predators. Not to be underestimated is the return to humans, as this species is edible. Understanding the behaviour of cadmium in aquatic systems, its effects at different trophic levels and the potential risks to human health from the consumption of contaminated seafood would therefore be essential for informed environmental management and policy decisions.

Saccharomyces cerevisiae supplemented diets mitigate the effects of waterborne cadmium toxicity on gilthead seabream (Sparus aurata L.): growth performance, haemato-biochemical, stress biomarkers, and histopathological investigations.

Yeast, Saccharomyces cerevisiae, has been utilized as a probiotic in aqua-feeds to promote growth and alleviate the stress in aquatic animals. On the other hand, cadmium (Cd) toxicity causes serious retardation of growth and welfare status of aquatic animals. The present study was conducted to evaluate the protective role of dietary yeast in mitigating the waterborne Cd toxicity effects on the growth, haemato-biochemical, stress biomarkers, and histopathological investigations of gilthead seabream (Sparus aurata L.). In a 3 × 3 factorial design, the acclimated fish (20-24 g) were randomly distributed into nine treatments in triplicates where they were fed on 0.0% (control), 0.5%, and 1.0% of yeast along with exposure to 0.0, 1.0, and 2.0 mg Cd/L for 60 days. All growth parameters and mRNA expressions of IGF-1 and GH genes as well as haematological parameters were markedly increased with the increase of dietary yeast levels; meanwhile these variables were significantly retarded with Cd exposure. Contradictory effects on the above-mentioned variables were observed with Cd toxicity. In contrast, blood cortisol, glucose, total cholesterol, and triglyceride, lactate dehydrogenase, alanine transaminase, aspartate transaminase, alkaline phosphatase, in addition to DNA fragments % were noticeably increased with Cd toxicity especially at the treatment of 2.0 mg Cd/L, while decreasing with increasing dietary yeast levels. Compared with the control fish group, Cd concentrations in the gill, liver, and muscle tissues of gilthead seabream were higher in Cd-exposed treatments, especially at the treatment of 2.0 mg Cd/L. Deposition of Cd in fish liver was higher than that in gill tissues but lowest Cd residue was observed in muscle tissues. No significant changes in Cd residues in fish organs were observed in yeast-fed fish with no Cd exposure. The Cd exposure negatively affected histological status of gill, liver, and kidney tissues of S. aurata; while feeding Cd-exposed fish on yeast diets lowered the Cd residues in fish organs and recovered the adverse effects of Cd toxicity. Hence, this study recommends the addition of bakery yeast (1.0%) to fish diets to improve the performance, overall welfare, and histopathological status of gilthead seabream, S. aurata.

Comparative neurotoxicity of dietary methylmercury and waterborne inorganic mercury in fish: Evidence of optic tectum vulnerability through morphometric and histopathological assessments.

This work investigated the effects of inorganic mercury (iHg) and methylmercury (MeHg) on the fish optic tectum morphology, viz. in relation to: (i) vulnerability of specific optic tectum layers; (ii) preferential targeting of Hg forms to neurons or glial cells; (iii) comparative toxicity of iHg and MeHg in this brain area that is in the maintenance of several fish behaviors. Two experiments exposing juvenile white seabream (Diplodus sargus) to waterborne iHg [HgCl2 (2 µg L-1)] and dietary MeHg (8.7 µg g-1) were performed, comprising both exposure (7 and 14 days; E7 and E14, respectively) and post-exposure (28 days; PE28) periods. Morphometric assessments were performed using stereological methods where the layers of the optic tectum were outlined, while its area and the number of neurons and glial cells were estimated. A histopathological assessment was also performed per section and per layer of optic tectum. iHg exposure did not trigger the loss of neurons during the exposure periods, while a decrease of glial cells was detected in a single layer of the optic tectum at E14. Differently, upon MeHg exposure, a decrease on the number of neurons and glial cells was found in several layers of optic tectum. In the post-exposure, both Hg forms triggered the loss of neurons, while only MeHg exposure led to a decrease on the number of glia cells. The histopathological assessment pointed out a higher toxicity of MeHg in the optic tectum layers, particularly in the post-exposure period, while no significant alterations were found in fish exposed to iHg. Hg forms targeted preferentially neurons. iHg and MeHg are relevant neurotoxicants to fish, with MeHg exposure leading to a higher toxicity than iHg in the optic tectum. After 28 days of post-exposure, iHg and MeHg neurotoxicity remained prominent, suggesting long-term effects of these toxicants.

Implication of adipocytes from subcutaneous adipose tissue and fatty acids in skin inflammation caused by λ-carrageenin in gilthead seabream (Sparusaurata).

The role of subcutaneous adipose tissue adipocytes and the effects of fatty acids on carrageenan-induced skin inflammation in gilthead seabream (Sparus aurata) were studied. Fish were injected intramuscularly with phosphate-buffered saline (control) or λ-carrageenin (1%), and skin samples collected at the injection site at 3 and 6 h post-injection (p.i.) were processed for histological study. In addition, the presence and levels of lipid classes, fatty acid methyl esters (FAME) and eicosanoids were evaluated in the skin samples obtained from the injected areas. Histological results indicated an increase in adipocyte area in fish sampled at 3 h p.i. with λ-carrageenin compared to fish in the control group. Furthermore, the frequency of adipocytes between 4500 and 5000 µm2 was increased at 6 h in the λ-carrageenin group compared to the control group. Analysis of lipid classes found that fish injected with λ-carrageenan showed increased free fatty acid (FFA) and sphingomyelin content at 3 and 6 h, respectively, compared to the control group. An increase in saturated fatty acids (SFA), n-6 polyunsaturated fatty acids (PUFA), and a decrease in the values of monounsaturated fatty acids (MUFA), n-3 PUFA and minor fatty acids were observed in fish skin at 6 h after λ-carrageenin injection, with respect to the values obtained in the control group. Regarding the analysis of eicosanoids, an increase in hydroxyeicosatetraenoic acid (5-HETE) was detected in the skin of fish at 6 h post-carrageenin injection compared to the control group. The presented results indicate the contribution of adipocytes and fatty acids in the development and regulation of the inflammatory response triggered by λ-carrageenin in gilthead seabream skin.

Role of Ion Channels in the Maintenance of Sperm Motility and Swimming Behavior in a Marine Teleost.

In oviparous marine fishes, the hyperosmotic induction of sperm motility in seawater (SW) is well established, however, the potential function of ion channels in the maintenance of post activated spermatozoon swimming performance remains largely unknown. Here, we investigated the influence of ion channels on the spermatozoon swimming parameters using the gilthead seabream (Sparus aurata) as a model for modern marine teleosts. Our data show that the SW-induced activation of seabream sperm motility requires three concomitant processes, the hyperosmotic shock, an ion-flux independent increase of the intracellular concentration of Ca2+ ([Ca2+]i), but not of [K+]i or [Na+]i, and the alkalization of the cytosol. The combination of all three processes is obligatory to trigger flagellar beating. However, the time-course monitoring of sperm motion kinetics and changes in the [Ca2+]i, [K+]i and [Na+]i in SW or in non-ionic activation media, showed that the post activated maintenance of spermatozoa motility is dependent on extracellular Ca2+ and K+. A meta-analysis of a seabream sperm transcriptome uncovered the expression of multiple ion channels, some of which were immunolocalized in the head and/or tail of the spermatozoon. Selective pharmacological inhibition of these ion channel families impaired the long-term motility, progressivity, and velocity of SW-activated spermatozoa. The data further revealed that some antagonists of K+-selective or Ca2+-selective channels, as well as of stretch-activated and mechanosensitive channels, altered the trajectory of spermatozoa, suggesting that these ion channels are likely involved in the control of the swimming pattern of the post activated spermatozoon. These combined findings provide new insight into the signaling pathways regulating spermatozoon activation and swimming performance in marine fishes.

Determination of very long-chain polyunsaturated fatty acids from 24 to 44 carbons in eye, brain and gonads of wild and cultured gilthead sea bream (Sparus aurata).

Very long-chain (> C24) polyunsaturated fatty acids (VLC-PUFA) play an important role in the development of nervous system, retinal function and reproductive processes in vertebrates. Their presence in very small amounts in specific lipid classes, the lack of reference standards and their late elution in chromatographic analyses render their identification and, most important, their quantification, still a challenge. Consequently, a sensitive and feasible analytical methodology is needed. In this work, we have studied the effect of chain length, as well as the number and position of unsaturations (or double bonds) on the response of GC-APCI-(Q)TOF MS, to establish an analytical method for VLC-PUFA quantification. The developed methodology allows the quantification of these compounds down to 2.5 × 10-3 pmol/mg lipid. The reduction of VLC-PUFA levels in lipid fractions of the organs from the herein sampled farmed fish suggesting a yet undetected effect on these compounds of high vegetable oil aquafeed formulations, that currently dominate the market.

Haematology reference values for Dicentrarchus labrax and Sparus aurata: A systematic review and meta-analysis.

Haematological parameters are frequently used as physiological indicators in aquaculture studies. These parameters also have extended applications in clinical evaluation, diagnosis and prognosis in fish health status. However, no normal reference range of values has been demonstrated in depth for any of these haematological parameters for the European sea bass (Dicentrarchus labrax) or gilthead seabream (Sparus aurata). The main objective of this article is to present for the first time through extended literature review, the haematological parameters normal range values for the two most important aquaculture fish species farmed in Mediterranean Sea, D. labrax and S. aurata, and to demonstrate their similarities and their differences. In this article, we also discuss the environmental and external factors affecting their normal blood parameters values and we propose fundamental guidelines on the reporting units.

Ultrasonography and X-ray micro-computed tomography characterization of the effects caused by carrageenin in the muscle of gilthead seabream (Sparus aurata).

The current work aimed to carry out an in vivo study of the λ-carrageenin-induced inflammation in the skin of gilthead seabream (Sparus aurata). The fish were injected intramuscularly with phosphate-buffered saline (PBS, as control) or λ-carrageenin (1% in PBS), and the injection zone was evaluated by real-time ultrasonography (Vevo Lab, VisualSonics) at 1.5, 3, 6, 12, and 24 h post-injection (p.i.). Results demonstrated that the skin thickness was increased in fish injected with λ-carrageenin and sampled at 1.5, 3, and 6 h p.i. However, the skin thickness of the injected area decreased to the normal values in those fish sampled at 12 and 24 h p.i. In addition, fish injected with λ-carrageenin and analysed at 1.5, 3, and 6 h p.i. showed, in the underlying muscle at the injection place, several hyperechoic small foci surrounded by an anechoic area which were not observed in control fish. Furthermore, the fish were analysed by X-ray micro-computed tomography (micro-CT). The analysis of the micro-CT acquisitions revealed also a dark area in the place of the injection with λ-carrageenin at 1.5, 3, and 6 h. These areas were smaller in fish analysed at longer times (12 h p.i.) and were almost disappeared in fish sampled at 24 h p.i. These areas had an average density of -850 to -115 HU, which did not correspond with any tissue density of the rest of the body. Furthermore, similar dark areas at the injection zones were never observed in control fish. Present results support the use of both non-invasive techniques to study the inflammatory process in fish of commercial interest such as gilthead seabream.

Ex vivo exposure to titanium dioxide and silver nanoparticles mildly affect sperm of gilthead seabream (Sparus aurata) - A multiparameter spermiotoxicity approach.

Nanoparticles (NP) are potentially reprotoxic, which may compromise the success of populations. However, the reprotoxicity of NP is still scarcely addressed in marine fish. Therefore, we evaluated the impacts of environmentally relevant and supra environmental concentrations of titanium dioxide (TiO2: 10 to 10,000 µg·L-1) and silver NP (Ag: 0.25 to 250 µg·L-1) on the sperm of gilthead seabream (Sparus aurata). We performed short-term direct exposures (ex vivo) and evaluated sperm motility, head morphometry, mitochondrial function, antioxidant responses and DNA integrity. No alteration in sperm motility (except for supra environmental Ag NP concentration), head morphometry, mitochondrial function, and DNA integrity occurred. However, depletion of all antioxidants occurred after exposure to TiO2 NP, whereas SOD decreased after exposure to Ag NP (lowest and intermediate concentration). Considering our results, the decrease in antioxidants did not indicate vulnerability towards oxidative stress. TiO2 NP and Ag NP induced low spermiotoxicity, without proven relevant ecological impacts.

The Effect of 17α-Ethynilestradiol and GPER1 Activation on Body and Muscle Growth, Muscle Composition and Growth-Related Gene Expression of Gilthead Seabream, Sparus aurata L.

Endocrine-disrupting chemicals include natural and synthetic estrogens, such as 17α-ethynilestradiol (EE2), which can affect reproduction, growth and immunity. Estrogen signalling is mediated by nuclear or membrane estrogen receptors, such as the new G-protein-coupled estrogen receptor 1 (GPER1). The present work studies the effect of EE2 and G1 (an agonist of GPER1) on body and muscle parameters and growth-related genes of 54 two-year-old seabreams. The fish were fed a diet containing EE2 (EE2 group) and G1 (G1 group) for 45 days and then a diet without EE2 or G1 for 122 days. An untreated control group was also studied. At 45 days, the shortest body length was observed in the G1 group, while 79 and 122 days after the cessation of treatments, the shortest body growth was observed in the EE2 group. Hypertrophy of white fibers was higher in the EE2 and G1 groups than it was in the control group, whereas the opposite was the case with respect to hyperplasia. Textural hardness showed a negative correlation with the size of white fibers. At the end of the experiment, all fish analyzed in the EE2 group showed a predominance of the gonadal ovarian area. In addition, the highest expression of the mafbx gene (upregulated in catabolic signals) and mstn2 (myogenesis negative regulator) was found in EE2-exposed fish.

Effect of partial substitution of fishmeal with insect meal (Hermetia illucens) on gut neuromuscular function in Gilthead sea bream (Sparus aurata).

Alternative nutrient sources to fishmeal for fish feed, such as insect meals, represent a promising sustainable supply. However, the consequences for fish digestive function have not been exhaustively investigated. In the present study we evaluated the effect of partial fishmeal substitution with 10% Hermetia illucens (Hi10) larvae meal on the neuromuscular function of proximal and distal intestine in gilthead sea bream. In animals fed with insect meal, weight and growth parameters were similar to controls fed with conventional fishmeal. In addition, no anomalies in intestinal gross morphology and no overt signs of inflammation were observed. The gastrointestinal transit was significantly reduced in Hi10 fed animals. In the proximal and distal intestine longitudinal muscle, Hi10 feeding downregulated the excitatory cholinergic and serotoninergic transmission. Sodium nitroprusside-induced inhibitory relaxations increased in the proximal intestine and decreased in the distal intestine after Hi10 meal. Changes in the excitatory and inhibitory components of peristalsis were associated with adaptive changes in the chemical coding of both proximal and distal intestine myenteric plexus. However, these neuromuscular function alterations were not associated with considerable variations in morphometric growth parameters, suggesting that 10% Hi meal may represent a tolerable alternative protein source for gilthead sea bream diets.

Hypoosmotic stress induced functional alternations of intestinal barrier integrity, inflammatory reactions, and neurotransmission along gut-brain axis in the yellowfin seabream (Acanthopagrus latus).

The gut-brain axis plays a major role in multiple metabolic regulation processes, but studies regarding its responses to environmental stress in fish are still limited. In this study, we performed transcriptome sequencing analysis and enzyme-linked immunosorbent assay (ELISA) in yellowfin seabream (Acanthopagrus latus) exposed to environments with different water salinity (freshwater: 0 ppt; low-saline water: 3 ppt; brackish water: 6 ppt). According to transcriptome analysis, 707 and 1477 genes were identified as differentially expressed genes (DEGs) between freshwater and brackish water treatments in the brain and gut, respectively. Brain DEGs were significantly enriched into a set of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with signal transduction, most of which were downregulated. Gut DEGs were enriched into a neurotransmission-relevant KEGG pathway tryptophan metabolism, and the downregulated DEGs were enriched into the KEGG pathway focal adhesion. ELISA demonstrated significant physiological responses of the brain and gut across treatments, as determined by the concentrations of tight junction protein ZO-2, interleukin 1ß, and serotonin. Under hypoosmotic stress, the functions of the gut-brain axis are altered via impairment of intestinal barrier integrity, by disturbance of gut-brain neurotransmission, and through tissue-damaging inflammatory reactions. Our work identified candidate genes which showed significantly differential expression in the gut-brain axis when yellowfin seabream encountered hypoosmotic stress, which could shed lights on the understanding of the potential osmotic regulation mechanisms of the gut-brain axis in teleost.

Lack of negative effects of fasting of gilthead seabream (Sparus aurata) breeders during the spawning period on maternal and egg nutrient composition, fertilization success, and early embryo/larval development.

The effect of fasting on spawning performance, maternal, and egg nutrient composition, and on embryo/larval development was monitored in gilthead seabream (Sparus aurata). Two broodstocks were fasted during two consecutive years, for a period of 43 and 54 days within the spawning season, in a preliminary (year 1, 5-year-old breeders) and the main study (year 2, 6-year-old breeders), respectively. Mean daily fecundity showed a declining trend during fasting in the main study only, while fertilization success was high in both years and it was not affected by fasting, as was hatching and 5-day larval survival. There was a loss of 23.5% of maternal body mass due to fasting, and a reduction in gonadosomatic and hepatosomatic indexes, as well as crude protein in maternal muscle and gonads, but not in the liver. After fasting, muscle Σω-6 PUFA and C18:3ω-3 were reduced while C20:4ω-6, 20:5ω-3/20:4ω-6, and C22:6ω-3/20:4ω-6 increased; in the liver, significant reductions were observed in C16:0, C18:3 ω-3, 20:5ω-3/C22:6ω-3 and increases in C18:0, C20:5ω-3, Σω-6 PUFA, and 20:5ω-3/20:4ω-6; in gonads, C15:0, ΣMUFA, 20:5ω-3/C22:6ω-3, 20:5ω-3/20:4ω-6 were increased, while C18:1ω-9 and C20:5ω-3 decreased. Contrary to maternal tissues, the energy density and proximate composition of the eggs did not change due to fasting. The study suggests that fasting of gilthead seabream breeders for 6-8 weeks during the spawning period does not affect spawning performance, egg proximate composition, or embryo and early larval development since maternal nutrient reserves are mobilized to maintain optimal egg nutrient composition.

Establishment and characterisation of single cell-derived embryonic stem cell lines from the gilthead seabream, Sparus aurata.

An important bottleneck in fish aquaculture research is the supply and maintenance of embryos, larvae, juvenile and adult specimens. In this context, cell lines represent alternative experimental models for in vitro studies that complement in vivo assays. This allows us to perform easier experimental design and sampling and avoid the sacrifice of animals. Embryonic stem (ES) cell lines have attracted increasing attention because they have the capability to proliferate indefinitely and could be differentiated into any cell type of the organism. To minimise cell heterogeneity and increase uniformity of in vitro studies results, in this manuscript we report the development and characterisation of two single cell-derived ES cell lines (monoclonal) from the morula stage embryos of the gilthead seabream, Sparus aurata, named as SAEC-A3 and SAEC-H7. Both cell lines have been passaged for over 100 times, indicating the establishment of long-term, immortalised ES cell cultures. Sequence analyses confirmed the seabream origin of the cell lines, and growth analyses evidenced their high viability and proliferating activity, particularly in culture medium supplemented with 10-15% fetal bovine serum and 22 °C. Both cell lines showed the ability to generate embryoid bodies and show different sensitivity and response to all-trans retinoic acid. The analysis of epithelial (col1α1) and neuronal (sox3) markers in differentiated cultures revealed that SAEC-A3 tended to differentiate towards epithelial-like cells whereas SAEC-H7 tended to differentiate towards neuronal-like cells. Both cell lines were efficiently transfected with pDsRed2-ER and/or pEGFP-N1 plasmids, indicating that they could represent useful biotechnological tools. Daily expression of pcna showed significant expression rhythms, with maximum levels of cell proliferation during the day-night transition. Currently, these cell lines are being successfully used as experimental models for the study of cellular metabolism, physiology and rhythms as well as for toxicological, pharmacological and gene expression analyses.

Relation of gilthead seabream (Sparus aurata) seasonal reproductive activity to hematology, serum biochemistry, histopathology, and Brdt gene expression.

This study aimed to find the relation of Sparus aurata (gilthead seabream) reproductive activities to some blood parameters as complete blood count, liver enzymes, some hormones related to reproduction process and microscopic findings of gonads, as well as expression of Bromodomain testis-specific gene. Eighty-eight sexually mature seabream were collected and investigated through the four seasons. Red blood cells were higher in autumn and spring. Hemoglobin was high in summer, MCV highest values ​​were seen in winter and summer, while MCHC was highest in summer. The values ​​of white blood cells increased significantly in spring, summer, and autumn compared with winter. The highest value of lymphocytes was recorded in spring and autumn. Eosinophil was recorded the highest value in the spring. The highest value of segmented neutrophils was recorded in summer. The highest value of band neutrophil was recorded in summer and winter. Alanine aminotransferase and aspartate aminotransferase showed high values in the winter. Luteinizing hormone (LH) was higher in females, males, and hermaphrodites during winter. Follicle-stimulating hormone (FSH) was higher in females during spring. The highest value of estradiol 17-ß and progesterone was recorded in summer. The highest value of total testosterone was recorded in spring. Microscopically, ovaries were immature and inactive during spring and summer but well developed in autumn and winter. During spring and summer, testes were immature and began spermatogenesis process but well developed with the appearance of spermatids and spermatozoa during autumn and winter. The expression of Brdt was higher in testes than ovary. Brdt recorded high expression in autumn and spring than in summer and winter.

Long lasting effects of early temperature exposure on the swimming performance and skeleton development of metamorphosing Gilthead seabream (Sparus aurata L.) larvae.

Temperatures experienced during early ontogeny significantly influence fish phenotypes, with clear consequences for the wild and reared stocks. We examined the effect of temperature (17, 20, or 23 °C) during the short embryonic and yolk-sac larval period, on the swimming performance and skeleton of metamorphosing Gilthead seabream larvae. In the following ontogenetic period, all fish were subjected to common temperature (20 °C). The critical swimming speed of metamorphosing larvae was significantly decreased from 9.7 ± 0.6 TL/s (total length per second) at 17 °C developmental temperature (DT) to 8.7 ± 0.6 and 8.8 ± 0.7 TL/s at 20 and 23 °C DT respectively (p < 0.05). Swimming performance was significantly correlated with fish body shape (p < 0.05). Compared with the rest groups, fish of 17 °C DT presented a slender body shape, longer caudal peduncle, terminal mouth and ventrally transposed pectoral fins. Moreover, DT significantly affected the relative depth of heart ventricle (VD/TL, p < 0.05), which was comparatively increased at 17 °C DT. Finally, the incidence of caudal-fin abnormalities significantly decreased (p < 0.05) with the increase of DT. To our knowledge, this is the first evidence for the significant effect of DT during the short embryonic and yolk-sac larval period on the swimming performance of the later stages.

Ultrasonography study of the skin wound healing process in gilthead seabream (Sparus aurata).

This work aimed to carry out an in vivo study of the skin healing process in gilthead seabream (Sparus aurata) after being experimentally wounded. Firstly, the structure of normal skin was studied by real-time ultrasonography (Vevo Lab, VisualSonics) and light microscopy. Besides this, experimental wounds were made on the left flank of each fish with a circular biopsy punch (8 mm diameter) below the lateral line. The healing process was assessed on live fish at 0, 6, 11 and 23 days post-wounding using the real-time ultrasonography in B-mode and Power Doppler mode (Vevo 3100 FUJIFILM, VisualSonics). Through the ultrasonography images, both the skin structure and the evolution of the changes that wounds originated in the surrounding tissues were studied in vivo over time. Concomitantly, the pattern of neovascularization in the wounded area was followed during the healing process and it was demonstrated that, although the neovascularization started very early after the skin damage, it was increased in wounded areas from day 11 post-wounding onwards. The results obtained proved the utility and power of using ultrasounds in fish to evaluate in vivo complex biological processes in real time, which are difficult to study by other methodologies. The present data shed some light on the reparation of external injuries in aquatic vertebrates.

Strengths and weaknesses in the long-term sustainability of two sympatric seabreams (Argyrops spinifer and Rhabdosargus haffara, Sparidae).

Argyrops spinifer and Rhabdosargus haffara are two sympatric seabream species making important contributions to fisheries landings in the western Arabian/Persian Gulf. We identified the strengths and weaknesses in the long-term sustainability of A. spinifer and R. haffara stocks by integrating multiple sources of data, including fisheries catch and effort statistics, life history traits, scientific trawl surveys and historical length frequency distribution. Four strengths were identified in A. spinifer: wide distribution of juveniles, positive association to the network of de facto fishing exclusion areas created by hundreds of oil-gas facilities, early maturation and the existence of large and old individuals. A. spinifer suffers from two potential weaknesses: slow growth rate and higher exploitation pressure on the small-sized individuals. R. haffara, on the other hand, has a strength of having a short life span and a fast growth rate, characteristics that make it robust to unfavourable conditions. R. haffara suffers from two weaknesses: the lack of association to the oil and gas facilities, and the preference for nearshore shallow waters with stronger negative anthropogenic impacts. Identified strengths and weaknesses of these two sparids provided a preliminary assessment about their long-term sustainability, as well as a roadmap about how to develop different management strategies to meet specific objectives.

Experimental evidence of physiological and behavioral effects of microplastic ingestion in Sparus aurata.

Increasing global research has identified microplastics (MPs) to be impacting marine organisms. The present work aimed at investigating the physiological and behavioral effects of thirty-six juvenile Sparus aurata exposed to control, virgin and weathered MPs enriched diets during a 21-day period under controlled conditions. Physiological effects were assessed in liver and brain using the following biomarkers: activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GRd), the detoxifying enzyme glutathione S-transferase (GST) and malondialdehyde (MDA) as indicative of lipid peroxidation. Individuals were recorded for behavior analysis (i.e. social interactions and feeding behavior). Results revealed an increase in cellular stress from control to weathered fish groups, with the virgin group showing intermediate levels in all quantified biomarkers. Significant differences were found in the liver for all biomarkers except for MDA, suggesting that exposure time to MPs in this experiment is long enough to trigger the activation of antioxidant enzymes but not to produce cell damage by lipid peroxidation. In brain tissue samples, fish from the weathered group presented significantly higher values for CAT and SOD, highlighting its function as primary antioxidants. Regarding behavioral effects, results showed that the two MPs enriched groups were significantly bolder during social interactions and, although no significantly, tended to be more active during feeding. In conclusion, MPs which have been weathered in marine environmental conditions produces a higher physiological response than virgin MPs but also, a physiological response is variable depending on the tissue analyzed. In addition, a short period to MP exposure seems to affect overall social and feeding behavior but, further research is needed to assess long-term effects of MP ingestion and its potential consequences on fish populations.

Oxygen uptake, heart rate and activities of locomotor muscles during a critical swimming speed protocol in the gilthead sea bream Sparus aurata.

Oxygen uptake, heart rate and contraction frequencies of slow oxidative (SO) and fast glycolytic (FG) muscle were measured simultaneously in gilthead seabream Sparus aurata submitted to stepwise increases in current speed in a swimming respirometer. Variation in oxygen uptake was closely related to variation in heart rate, over initial steps these rose in concert with an increase in contraction frequency of SO muscle. There was an asymptote in oxygen uptake and heart rate at high speeds that reflected a transition from exclusive use of aerobic SO muscle to a combination of SO and anaerobic FG muscle, and which preceded fatigue.